Metabolic Analysis On Ophiocordyceps Sinensis Infection Of Hepiaua Larva Indoor

Ophiocordyceps sinensis is a complex of Hepiaua larva and the parasitic fungi Hirsutella sinensis,and is an endangered species in traditional Chinese medicine.There have been many reports on the metabolism of Ophiocordyceps sinensis and its anamorphic strains(Hirsutella sinensis).However,there have been few reports on the metabolic changes after the artificial inoculation of the Hepiaua larvae by the fungi Hirsutella sinensis.In this study,the cultured Hepiaua larva were infected respectively with the mycelium of Hirsutella sinensis and the ascospores of Ophiocordyceps sinensis indoor,and the infection effect and metabolic changes were analyzed.At the same time,the metabolites of the rigid worm and natural Ophiocordyceps sinensis were compared and analyzed to find out the metabolic differences between the artificially infected rigid worm and the natural Ophiocordyceps sinensis and provide a theoretical basis for the cultivation and utilization of the artificial Cordyceps.In this study,we first compared the differences in infection between the ascospores of natural Ophiocordyceps sinensis and the anamorphic strains of Ophiocordyceps sinensis(Hirsutella sinensis)on the Hepiaua larva.The results showed that the infection of Hepiaua larva with ascospores suspension can achieve better infection effect.The principal component analysis(PCA)of the metabolites obtained from the rigid worms obtained by different inoculum infestations,natural Ophiocordyceps sinensis,the cultured mycelium of Hirsutella sinensis and the natural rigid worms collected from the wild,It was found that the rigid worms obtained from ascospores infestation were most similar to the natural rigid worms and natural Ophiocordyceps sinensis.However,the results of heat map analysis(HCA)showed that there was a certain difference between the rigid worms obtained by artificial infestation and natural Ophiocordyceps sinensis.The major differential metabolites of the the rigid worms and natural Ophiocordyceps sinensis include malic acid,deoxyadenosine,methyl guanosine,α-linolenic acid,PE,PC and other Glycerophospholipid,Hericene B,ceramide,glucopyranoside,peptides,palmitoyl serine phosphate,cycloserine,L-valine,L-arginine,phenylasoleucine,glycerophosphocholine,phytosphingosine-1-phosphate,pyrrolidine and so on.From this study,the difference in these metabolites does not necessarily mean that the artificially infected worms are not as good as the natural Ophiocordyceps sinensis.Studies have shown that the content of some beneficial metabolites of the worms obtained by artificial infestation is higher than that of natural Ophiocordyceps sinensis,such as malic acid,ceramide,nucleosides,α-linolenic acid,PE,PC and other metabolites,indicating that some worms obtained by artificial infections may have a higher nutritional health value.The results of full-spectrum mass spectrometry analysis of the worms successfully infected by ascospores suspension with the natural Ophiocordyceps sinensis,it was found that most of the samples in the group of worms were very similar to the metabolites of natural Ophiocordyceps sinensis.Therefore,these worms may be used as substitutes for natural Ophiocordyceps sinensis.Of course,some samples in the group of worms have also been found to contain certain unique compounds such as yeastine,cytochalasin C,Aranorosinol A,paecilosetin,farinosone A.By referring to the relevant literature,we found that these substances belong to some typical secondary metabolites of insect-producing fungus associated with Cordyceps,such as paecilosetin produced by Paecilomyces farinosus.This phenomenon indicates that these worms were once infected by the fungi Hirsutella sinensis and other fungi,resulting in many different metabolites.This also explains why there is a large difference in metabolites between the worms infected by ascospores suspension and the natural Ophiocordyceps sinensis in the heat map.High-resolution LC/MS analysis of the components showed that these worms could not be used as a substitute for natural Ophiocordyceps sinensis.In this study,inoculation experiments revealed that the Hepiaua larva successfully infected with ascospores suspension have potential as substitutes for natural Ophiocordyceps sinensis.High-resolution liquid chromatography and mass spectrometry can accurately determine the compositional differences between artificial cultures and natural Ophiocordyceps sinensis to identify the cultures or dead worms that are consistent with natural Ophiocordyceps sinensis in the components,while avoiding samples of harmful bacterial infections to replace natural Ophiocordyceps sinensis.In order to expand the source of cordyceps medicine or find more safe alternatives to create greater economic benefits,it is also conducive to the protection of wild Cordyceps resources and ecological environment.This study also showed that artificial inoculation of Hirsutella sinensis on Hepiaua larva is more likely to contaminate the bacteria.Especially in mixed infections,it is difficult to judge from morphology.If there is no further analysis and use it as a substitute for Ophiocordyceps sinensis sightless,there may be potential safety trouble.In the actual operation of large-scale artificial cultivation of Ophiocordyceps sinensis and its substitutes,there is still a need for further in-depth research on how to avoid the contamination of germs to ensure the effectiveness and safety of the products.